Amniotic membrane and amniotic fluid-derived cells - Future Medicine

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Amniotic membrane and amniotic fluid-derived cells: potential tools for regenerative medicine? Human amniotic membranes and amniotic fluid have attracted increasing attention in recent years as a possible reserve of stem cells that may be useful for clinical application in regenerative medicine. Many studies have been conducted to date in terms of the differentiation potential of these cells, with several reports demonstrating that cells from both the amniotic fluid and membrane display high plasticity. In addition, cells from the amniotic membrane have also been shown to display immunomodulatory characteristics both in vivo and in vitro, which could make them useful in an allotransplantation setting. Here, we provide an overview comparing the latest findings regarding the stem characteristics of cells from both the amniotic membrane and amniotic fluid, as well as on the potential utility of these cells for future clinical application in regenerative medicine. keywords: amnion n amniotic fluid n clinical application n placenta n stem cells

In the field of regenerative medicine, stem cells represent a useful tool for maintaining or restoring the function of damaged or diseased tissues and organs. Stem cells are typically classified according to their ability to differentiate toward different cell types. Totipotent cells of the morula are able to differentiate into all cell types of an organism, including cells of the extraembryonic tissues. Pluripotent stem cells are able to form all cell types of all three germ layers (endoderm, mesoderm and ectoderm), although they are unable to form cell types of the extraembryonic tissues. Pluripotency is typically associated with embryonic stem cells, which are characterized by the expression of particular markers, including octamer-binding protein (OCT)-4, stage-specific embryonic antigen (SSEA)-3, SSEA-4, tumor rejection antigen (TRA)-1–60, TRA-1–81, Nanog, reduced expressin (Rex)-1 and SRY-related HMG-box gene 2 (SOX-2) [1–4] . Multipotent cells are capable of more limited differentiation compared with pluripotent cells, and are generally able to form cell types of just one particular germ layer. Finally, progenitor cells are already partially committed in terms of their differentiation potential, and are only able to form one or a few cell types within a particular lineage. The functional features of mouse embryonic stem cells are often used as a benchmark for the defining properties of pluripotent cells. Specifically, such cells should: Be able to be cultured indefinitely in the undifferentiated state;

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Be clonally derived as well as capable of differentiation toward all three germ layers;

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Be able to form teratomas in vivo;

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Be capable of giving rise to any cell type in the body after colonization of a host blastocyst;

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Be stably diploid and maintain a normal karyotype in vitro [4] .

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Although much progress has been made in characterizing different types of stem cells to date, several limitations exist in the potential clinical applicability of these cells. For example, although embryonic stem cells undoubtedly display a great deal of clinical potential owing to their high differentiation potential and ease of propagation, these cells are also associated with a high rate of tumor induction after transplantation [5,6] , and immune rejection of these cells remains a fundamental limitation to their clinical applicability [7] , while their use provokes ethical debate since the procurement of human embryonic stem cells requires destruction of the human embryo. Recently, the generation of induced pluripotent stem (iPS) cells from differentiated human somatic cells has been reported through retroviral transduction of key transcription factors [8,9] . Although these findings are very promising for the field of regenerative medicine because they provide the possibility of generating patient- or disease-specific pluripotent stem cells without the need for human embryos, the presence of transgenes in these cells could limit their therapeutic use. Regen. Med. (2009) 4(2), 275–291

Ornella Parolini1†, Maddalena Soncini2, Marco Evangelista2 & Dörthe Schmidt3 Author for correspondence: Centro di Ricerca E Menni, Fondazione Poliambulanza – Istituto Ospedaliero, Via Bissolati, 57, 25124 Brescia, Italy Tel.: +39 030 2455 754/705 Fax: +39 030 2455 704 E-mail: [email protected] 2 Centro di Ricerca E Menni, Fondazione Poliambulanza – Istituto Ospedaliero, Brescia, Italy 3 Regenerative Medicine Program, Clinic for Cardiovascular Surgery & Department of Surgical Research University & University Hospital Zurich, Switzerland †

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Parolini, Soncini, Evangelista & Schmidt

Multipotent adult stem cells represent an attractive alternative stem cell source for regenerative medicine and are currently used for a variety of therapeutic purposes [10] . These cells are capable of differentiation toward several lineages, with the advantage that they can be used in autologous therapies, thereby overc oming difficulties associated with immune rejection. However, these cells are often present in low numbers and can be difficult to maintain in culture, and they do not display the pluripotency of embryonic stem cells. In investigating other potential sources of adult stem cells, scientists have recently begun to dedicate attention to cells of the term placenta and amniotic fluid, envisaging that cells from both sources may demonstrate a high degree of plasticity. In addition, cells from the placenta are also readily available, while cells from the amniotic fluid offer the possibility of being utilized for autologous fetal or perinatal therapies. Study of the placenta as an alternative stem cell source is supported by the fact that placental tissues derive from pregastrulation embryonic cells, supporting the possibility that they may retain some of the plasticity that is characteristic of these cells. Furthermore, the fact that placental tissues contribute to maintenance of feto–maternal tolerance suggests that cells from these tissues may have immunomodulatory properties that would permit their use in an allotransplantation setting. Finally, since placental tissues are generally discarded after birth, the use of placental cells avoids the ethical problems associated with embryonic stem cells. Table 1 shows a comparison, in terms of key functional and phenotypic aspects, between cells derived from the amniotic membrane and amniotic fluid, and embryonic stem cells and bone marrow-derived mesenchymal stromal cells (BM-MSCs), which represent a well-characterized adult stem cell source. Human amniotic membrane The placenta is a feto–maternal organ that consists of two components: a maternal component, termed the decidua, which is derived from the endometrium, and a fetal component, which includes the amniotic membrane, the chorionic membrane and the chorionic plate, from which villi extend and make intimate contact with the uterine decidua during pregnancy. The amniotic membrane represents the innermost of the two fetal membranes that surround the fetus during pregnancy. This thin, avascular membrane, which lines the amniotic cavity and 276

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is bathed in amniotic fluid, is contiguous over the umbilical cord with the fetal skin. It is composed of the amniotic epithelium (AE), which is made up of an uninterrupted single layer of flat, cuboidal and columnar cells, and which in turn lies on the amniotic mesoderm (AM), in which a network of fibroblast-like mesenchymal cells and rare macrophages are widely dispersed. Embryologically, the two layers of the amniotic membrane originate before gastrulation. In particular, at day 8–9 after fertilization, once implantation of the blastocyst has occurred, the inner cell mass of the blastocyst differentiates into two layers, termed the epiblast and the hypoblast, that together form the bilaminar embryonic disc. From the epiblast, some small cells that will later constitute the AE appear between the wall of the blastocyst (the tropho blast) and the embryonic disc, and enclose a space that will become the amniotic cavity. The three germ layers (ectoderm, mesoderm and endoderm) also develop from the epiblast. From the other side of the bilaminar disc, some cells from the hypoblast migrate along the inner wall of the blastocyst cavity, giving rise to the exocelomic membrane, which together with trophoblastic cells, form the extraembryonic reticulum. Some hypoblast cells then migrate along the outer edges of extraembryonic reticulum to form a connective tissue known as the extraembryonic mesoderm, which surrounds the yolk sac and amniotic cavity, and later forms the AM, the chorionic mesoderm and the Wharton’s Jelly of the umbilical cord [11,12] .

Clinical application of the amniotic membrane The use of amniotic membrane as a therapeutic agent has been studied for decades. In 1910, Davis was the first to report the use of fetal membranes as surgical materials in skin transplantations [13] . Since then, it has been shown that the amniotic membrane has anti-inflammatory [14–16] , antifibroblastic [17] and antimicrobial [18] properties, and also possesses low immunogenicity [19,20] . Several other applications for amniotic membrane in surgery have since been reported, including its use as a biological dressing for treatment of skin wounds, burn injuries and chronic leg ulcers [21–28] , and in prevention of tissue adhesion in surgical procedures [29–31] . Transplantation of amniotic membrane was introduced to the field of ophthalmology in the 1940s for the treatment of ocular burns [32,33] . Since then, the amniotic membrane has been applied widely in ocular surface reconstruction, future science group

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27 [112]; more than 250 [104]

5–10 [41]

Not reported

Human amniotic mesenchymal stromal cells

Human amniotic From passage fluid-derived cells 3–26: 3.6 days [112] ; 36 h [104]

30 [45]

10 [157,158] ; 20 [159]

At passage 5: 24 h At passage 30: 18 h [45]

36–72 h [157]

Human amniotic epithelial cells

Human bone marrow-derived mesenchymal stromal cells

[70,99,104,110,111]

OCT-4, Rex-1, GATA4

[49,67,71–73]

OCT-4, SOX-2, Rex-1, Nanog

[42,44]

OCT-4, SOX-2, Nanog

[159]

OCT-4, SOX-2, Rex-1

OCT-4, SOX-2, Nanog [149] , Rex-1 [150] .

Differentiation potential

CD105, CD90, CD73, CD44, CD29, CD13, CD10, CD49c, CD49d, CD49e, CD54, CD166, CD271low, CD349, CD140, CD324, E-cadherin [41,48,49,68,69,72] CD105, CD90, CD73, CD166, CD49e, CD58, CD54, CD123, CD71, CD44, CD117, CD133, SSEA-4, CD29, stem cell factor, SH2, SH3, SH4, c-Met [99,100,104–107]

[42–44,46,48,49]

Expression of MHC class I [48,49] Inhibition of in vitro T‑cell proliferation [49,140] Long-tem engraftment in various organs after injection into neonatal swine and rats [139] Expression of MHC class I [100,104] Low [104] or negative [100] expression of MHC class II Contradictory reports regarding in vivo tolerance/rejection [104,105]

Endodermal, ectodermal and mesodermal differentiation [104]

In vitro differentiation toward endodermal, mesodermal and ectodermal lineages [41] Differentiation into cardiomyocytelike cells in vivo [71]

Endodermal, mesodermal and ectodermal differentiation in vitro [155] Contribution to all three germ layers in chimeric embryoid bodies [156] Teratoma formation in vivo [5] Expression of MHC class I, absence Differentiation in vitro toward at CD73, CD90, CD105, in of MHC class II [161] least osteo-, adipo- and the absence of CD14, chondrogenic lineages [163,164] CD34, CD45 [160] In vitro inhibition of T- and B-cell proliferation, as well as generation of Data also reported supporting monocyte-derived dendritic cells in vitro endodermal and ectodermal differentiation [163] [160,162] Conflicting results reported in clinical studies regarding prevention of graft versus host disease [163] CD105, CD90, CD73, Expression of MHC class I [50] In vitro differentiation toward CD44, CD29, CD13, CD10, Survival without signs of endodermal, mesodermal and CD166, CD49e, CD117, ectodermal lineages [41] immunological rejection after ABCG2, CD9, CD24, transplantation into spinal cordContribution to all three germ layers E-cadherin, integrins α6 injured bonnet monkeys [59] in chimeric embryoid bodies [45] and β1, SSEA-3, SSEA-4, Perform functions of hepatic [46,65,66] TRA-1–60, TRA-1–81 and pancreatic cells [67] in vivo

SSEA–3, SSEA-4, TRA-l-60, TRA-1‑81 [5]

Low expression of MHC class I, absence of MHC class II [151,152] Controversial data reported regarding in vivo immunomodulatory properties [7,153,154]

Indefinite [4]

16 h [147] ; 32 h

Human embryonic stem cells

[148]

Immunological features

Cell surface markers Cell doubling Maximum Expression of time numbers of pluripotencypassages associated transcription factors

Cell type

Table 1. Comparison of key features of human amniotic membane- and fluid-derived cells with two well-characterized human stem cell sources.

Amniotic membrane & amniotic fluid-derived cells

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where it is used as a substrate to promote development of normal corneal or conjunctival epithelium in the treatment of corneal erosions, central and peripheral ulcers and perforations, pterygium surgery, glaucoma surgery and keratopathy [34] . For these applications, amniotic membrane is usually preserved and stored using different methods such as lyophilization, cryopreservation, air-drying, irradiation or glycerol preservation [34,35] . Most of these methods, however, have been shown to result in a loss of cell viability either immediately or after a short period of storage [36–38] , suggesting that the biological effectiveness of the amniotic membrane may not always depend on the viability of its cells. With the aim of developing storage conditions that would retain the viability of these cells in cases where it is favourable to preserve their biological properties, Hennerbichler and co-workers have shown that amniotic membrane can be stored in L15-medium at room temperature or in Dulbecco’s Modified Eagle’s Medium at 37°C for up to 28 days with good retention of cell viability [39] . Recently, attempts to use viable amniotic cell monolayers in tissue engineering approaches without the use of scaffolds have been reported using the novel cell-sheet strategy, which has already been applied using other cell types [40] .

Human amniotic membrane-derived cells In addition to providing a useful substrate for therapeutic procedures, the amniotic membrane has more recently been investigated as a possible source of stem cells [41] . As mentioned above, the early origin of the amniotic membrane, which begins to develop before gastrulation occurs, supports the possibility that cells from this membrane maintain the plasticity of pregastrulation embryonic cells, and therefore some degree of stemness that may allow them to differentiate toward different cell lineages. Cells from the mesenchymal and epithelial regions of the amnion can be isolated easily by mechanical separation of the amnion from the chorion, followed by digestion with trypsin or dispase to release the epithelial cells, while the mesenchymal cells can be released through subsequent digestion with collagenase. When referring to cells derived from amniotic membrane, we will use the nomenclature established in a recent review: hAEC for human amniotic epithelial cells and hAMSC for human amniotic mesenchymal stromal cells [41] . 278

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Human amniotic epithelial cells Of the different cell types present in the amniotic membrane, hAECs have been investigated most extensively to date. Expression of embryonic stem cell markers such as SSEA-4, TRA1–60 and TRA-1–81 [42,43] , as well as expression of molecular markers of pluripotent stem cells, including OCT-4, SOX-2 and Nanog, have all been reported for hAECs [42,44] . The hypothesis that these cells may be pluripotent is supported by a study by Tamagawa et al., in which chimeric embryoid bodies were formed in vitro by mixing mouse embryonic stem cells with fluorescently labelled, clonally derived human amniotic cells. The resulting embryoid bodies were then cultured, resulting in the formation of primordial organs such as liver, lung, components of the digestive tract, skin, neural tube, spinal cord, blood vessels and blood cells, while fluorescence imaging revealed the presence of human cells in each of the organ primordia. Furthermore, the amniotic cells used in this study were shown to maintain a normal karyotype after multiple passages and could be cultured for more than 60 passages, after which active cell growth was still observed [45] . Epithelial markers such as cytokeratins are highly expressed on cultured hAECs, while mesenchymal markers are usually absent. Interestingly, however, appearance of the mesenchymal marker vimentin has been reported during culture of hAECs [20,46] . This may be due to spontaneous differentiation of these cells during culture, or to the so-called epithelial to mesenchymal transition, as also suggested by Sakuragawa and colleagues [47] . The potential of hAECs to differentiate in vitro toward cells of the mesodermal lineage has been demonstrated by several groups who have induced these cells to differentiate toward cells of the osteogenic, chondrogenic and adipogenic lineages [44,48,49] . HLA-A, -B and -C are expressed at very low levels on freshly isolated hAECs, but their expression increases during passages [50] . Other surface antigens present on hAECs include the ATP-binding cassette transporter G2 (ABCG2/BCRP), CD9, CD24, E-cadherin, integrins α6 and β1 and hepatocyte growth factor receptor (c‑Met). hAECs have been reported to express neuronal and glial markers [51], and also display neuronal functions such as synthesis and release of acetylcholine, catecholamines, neurotrophic factors, activin and noggin [47,52–56] , suggesting that these cells may have a predisposition to the neuronal lineage. Evidence that cells of the AE also provide support for in vitro growth future science group


of neuronal cell types comes from studies demonstrating that hAEC-conditioned medium has neurotrophic effects on rat cortical cells [55] and supports the survival of chicken neural retinal cells [57] . More recently, amniotic membrane has also been shown to sustain the growth and survival of chicken dorsal root ganglia neurons in the absence of neurotrophic factors [58] . Neuroprotective and neuroregenerative properties of hAECs are further supported by preclinical studies in animal models that demonstrate the utility of these cells in CNS regeneration during acute phases of injury. After transplantation of these cells into lesioned areas of a contusion model of spinal cord injury in monkeys, robust regeneration of host axons and enhanced survival of axotomized spinal cord neurons was observed, together with improved performance in locomotor tests [59] . Moreover, hAECs have also been shown to produce dopamine, prevent neuron degeneration and result in behavioural improvement after transplantation into a rat model of Parkinson’s disease [60–62] . Meanwhile, amelioration of behavioural dysfunction and reduced infarct volume have been observed after transplantation of hAECs into the brains of rats that had undergone middle cerebral artery occlusion [63] . To evaluate whether hAECs can be used as a cell source for transplantation to treat liver disease, the hepatocytic differentiation potential of these cells has also been investigated. Liverspecific transcription factors, including hepatocyte nuclear factor (HNF)-3γ and HNF-4α, CCAAT/enhancer-binding protein (CEBP)-α and -β and CYP450 enzymes, as well as hepatocyte-related genes including α1-antitrypsin (α1AT ), cytokeratin 18 (CK18), glutamine synthetase (GS), carbamoyl phosphate synthetase-1 (CPS-1), phosphoenolpyruvate carboxykinase (PEPCK ) and drug metabolism-related genes CYP2D6 and CYP3A4, have all been shown to be expressed by hAECs [42,64] . Albumin and α-fetoprotein (α-FP) production and typical hepatic functions such glycogen storage have also been described for hAECs in in vitro studies [65,66] . Meanwhile, studies in mice have demonstrated that hAECs are able to perform hepatic functions in vivo, with the observation that human α1AT could be detected by western blot in sera of severe combined immune deficiency (SCID) mice that had been transplanted with these cells [46] , while human albumin could be detected in the sera and peritoneal fluid of SCID mice that had received peritoneal implants of human amniotic membrane [66] . Furthemore, 2 weeks after transplantation of hAECs into future science group

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SCID mice, integrated α-FP- and albuminpositive cells could be detected in the hepatic parenchyma [65] . Together, these studies provide encouraging evidence that amniotic epithelial cells may be useful for restoring functionality of hepatic tissues that have been compromised by disease or injury. Pancreatic differentiation of hAECs has also been reported, with demonstration that these cells possess ultrastructural features characteristic of b-pancreatic cells, express the pancreatic marker amylase α2B (AMYB2) and produce glucagon after culture in pancreatic differentiation medium [44] . Wei and collegues have studied the in vivo functionality of hAECs that have been differentiated toward the pancreatic lineage [67] . In this study, pancreatic differentiation of hAECs was induced through culture in the presence of nicotinamide for 2–4 weeks. The resulting insulin-producing cells were then transplanted into streptozotocin-induced diabetic mice, and a subsequent normalization in blood glucose levels was observed [67] . Human amniotic mesenchymal stromal cells In addition to the strong data regarding the stem potential of hAECs that have been reported to date, several studies provide promising evidence that hAMSCs also harbour cells with stem characteristics. Several groups have shown that the phenotypic characteristics of hAMSCs are reminiscent of those described for BM-MSCs, with expression of typical mesenchymal markers (CD73, CD90 and CD105) in the absence of hematopoietic (CD34 and CD45) and monocytic (CD14) markers [41,48,49,68,69] . hAMSCs express low levels of HLA-ABC, but do not express HLA-DR, suggesting that these cells may be applicable in a clinical transplantation setting [48,49,70] . Pluripotency markers such as OCT-4 [49,67,71–73] , SOX-2, Rex-1 and Nanog [73] have been reported in hAMSCs, while positivity for the SSEA-3 or SSEA-4 in these cells is still debated [41,43,70] . Recently, a multipotent side population of cells was isolated by fluorescence-activated cell sorting from hAMSCs using the fluorescent dye Hoechst 33342. These cells displayed high expression of CD13, CD29, CD44, CD46, CD49b, CD49c, CD49e, CD59, CD140a and CD166, in the absence of CD34, CD45, CD49a, CD56, CD90, CD105, CD106 and CD117. In addition, these cells also displayed multilineage differentiation potential [74] , suggesting www.futuremedicine.com

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that selection based on Hoechst exclusion may constitute a method for selection of stem cells from the human amniotic mesenchymal region, although further studies would be required to validate this. Further evidence that hAMSCs may be pluripotent comes from several in vitro studies that describe differentiation of these cells toward lineages belonging to all three germ layers [41] . A predisposition of hAMSCs to neuronal differentiation has been demonstrated by the observation that these cells express neuronal (Nestin, Musashi 1, neuron-specific enolase, neurofilament medium, microtubule-associated protein [MAP]-2 and Neu-N) and glial markers (glial fibrillary acidic protein), with increased expression of some of these markers observed after culture of hAMSCs in specific neural-induction media [47,48,70,73] . Expression of hepatic markers such as albumin, α-FP, CK18, α1AT and HNF-4α has also been described for hAMSCs, with increased expression of these markers, together with production of α-FP and glycogen storage, reported after in vitro hepatic induction of these cells [75] . Investigation of the cardiomyogenic potential of hAMSCs has shown that these cells express the cardiac-specific transcription factor GATA4, as well as the cardiac-specific genes atrial myosin light chain (MLC)-2a, ventricular MLC-2v and the cardiac troponins cTn1 and cTnT. Moreover, integration of hAMSCs into cardiac tissue and differentiation of these cells into cardiomyocytelike cells has been observed after transplantation of hAMSCs into rat hearts following myocardial infarction [71] . Enhanced cardiomyogenic and vasculogenic differentiation of amniochorionic-derived cells was observed by Ventura and colleagues after exposure of these cells to a mixed ester of hyaluronan, butyric and retinoic acid (HBR), resulting in increased expression of cardiomyogenic (GATA4 and NKX2.5) and endothelial genes (VEGF and vWF), as well as cardiac proteins (sarco meric myosin heavy chain and α-sarcomeric actin) [76] . The angiogenic potential of amniotic membrane-derived cells has also been investigated, revealing that these cells basally express endothelial-specific markers (FLT-1 and KDR), and are capable of spontaneous differentiation into endothelial cells, which is enhanced through exposure to VEGF [72] . Finally, spontaneous differentiation of hAMSCs into myofibroblasts has also been observed after culture of these cells in standard medium. 280

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Interestingly, it has also been shown that the stromal surface of the amniotic membrane, as well as amniotic membrane stromal extract, are both able to reverse differentiated myofibroblasts back to fibroblasts, therefore suggesting that the stromal region of the amniotic membrane contains soluble factors that can regulate mesenchymal cell differentiation [77] . Amniotic membrane-derived cells from animal models Several reports have been published that explore the stem-like properties of amnion-derived cells obtained from animal models such as monkeys and rats, providing results that are in line with data obtained for human amnion-derived cells. The first of these studies is centered around monkey amniotic epithelial cells (MAECs), and is aimed at investigating whether these cells, like hAECs, may also have a neural predisposition, in order to assess whether MAECs could be useful in treating a monkey model of Parkinson’s disease. This study showed that MAECs possess the catecholamine-synthesizing enzymes and are also able to synthesize and release dopamine [78] . Furthermore, MAECs natively express both the D1 and D2-type dopamine receptor-binding sites, and are able to uptake dopamine in a time- and concentration-dependent fashion [79–81] . Cell-mediated therapy for the lysosomal storage disease mucopolysaccharidosis type VII has also been studied using MAECs that had been transduced with a recombinant adenovirus that caused them to express human b‑glucoronidase. A month after local intracerebral injection in monkeys, survival and widespread distribution of the transplanted cells was observed, suggesting that MAECs could indeed be useful in the treatment of lysosomal storage disorders that cause pathological abnormalities in the brain [82] . A neuronal predisposition of cultured rat amniotic epithelial cells has been demonstrated through the observation that these cells express neuronal markers such as neurofilament and MAP-2. Furthermore, after transplantation into the ischemic brains of mongolian gerbils, these cells were shown to migrate, survive and transform in a manner similar to neuronal and neural stem cells [83] . Rat amniotic epithelial cells have also been shown to produce neurotrophic factors such as neurotrophin (NT)-3 and BDNF and, when co-cultured with neural stem cells, were shown to provide a supportive environment for differentiation of neural stem cells into neurons [84] . future science group


Recently, a more comprehensive study encompassing isolation protocols, characterization and in vitro differentiation potential of stem cells derived from the rat amniotic membrane was reported by Marcus and colleagues [85] . In this study, amnion-derived stem cells (ADSCs) isolated from tissue explants of rat amniotic membrane (E 18.5) were shown to express the mesenchymal surface markers CD29 and CD90, but were negative for the lymphohematopoietic markers CD45 and CD11b. Moreover, gene expression analysis of cultured ADSCs revealed expression of transcription factors involved in maintaining pluripotency (Nanog, SOX-2), as well as genes that are characteristic of all three germ layers, including neuroectodermal structural proteins (MAP-2 and NF-M), mesodermal genes normally expressed in bone (osterix, alkaline phosphatase), fat (adipsin) and muscle cells (SM22-α), and endodermal genes of the lung and the liver (α1AT ). Pluripotency of these cells was further supported by in vitro differentiation of ADSCs and ADSC clonal lines toward cell lines belonging to all the three germ layers, as demonstrated by specific histochemical staining of the differentiated cells [85] . Furthermore, longterm survival of rat ADSCs, with no evidence of immunological rejection or tumor formation, has been observed after allogeneic in utero transplantation of these cells into the developing rodent brain [86] . Amniotic fluid The amniotic fluid is a protective and nourishing liquid that surrounds the fetus during pregnancy. As soon as the amniotic membrane has formed and lined the amniotic cavity, production of amniotic fluid begins, due mainly to active sodium and chloride transport across the amniochorionic membrane and fetal skin, with concomitant movement of water [87] . Later, in the second half of gestation, most of the fluid present is a result of micturation and secretion from the respiratory tract by the fetus itself [88] . In addition, the fetus also starts to swallow the amniotic fluid and to secrete it over the gastrointestinal tract [89] . Therefore, the composition of the amniotic fluid is subject to fluid dynamics and varies with gestational age [90,91] . Besides proteins, carbohydrates, fats, amino acids, enzymes, hormones and pigments, fetal cells can also be found in the amniotic fluid. Although amniotic fluid is routinely removed for prenatal diagnosis in a low-risk procedure (risk of miscarriage 1 out of 200) [92–94] using ultrasound-guided amniocentesis, only limited information exists future science group

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regarding the origin and characteristics of the cells present in this fluid. So far, various types of fetal cells have been found, including cells from all three germ layers [95–97] . Furthermore, besides fully differentiated cells, progenitor cells [98] and pluripotent embryonic-like stem cells have also been detected [99] . In order to better understand the origin and characteristics of this heterogeneous cell population, several studies have been performed using human amniotic fluid samples and animal models, with particular focus on the regenerative potential of these cells and the possibility of using them to develop cell-based therapies in the future.

Human amniotic fluid-derived cells It is well-known that amniotic fluid represents a very heterogeneous population that includes cells derived from the fetal membranes as well as from the fetus itself. Amniotic fluid-derived cells have long been classified on the basis of their morphological, biochemical and growth characteristics, with three main groups of colonyforming amniotic fluid-derived cells identified, namely, epithelioid-like cells, amniotic fluid-specific cells and fibroblast-like cells [95,97] . At the beginning of amniotic fluid-derived cell culture, both amniotic fluid-specific cells and epithelioid-like cells can be found, while fibroblast-like cells can only be cultured from some, but not all amniotic fluid samples, and usually appear late in the culture process. Amniotic fluid-specific cells persist during cell culture, whereas the levels of epithelioid-like cells decrease over time. It has been suggested that amniotic fluid-specific cells derive from fetal membranes and that epithelioid-like cells derive from fetal skin and urine, while fibroblast-like cells are thought to be derived from fibrous connective tissue and dermal fibroblasts [96,97] . Recently, however, it has been proposed that amniotic fluid-derived cells can be divided into two major categories, namely, adhering and nonadhering cells, and using a two-stage isolation and culture protocol [100] , it has been proposed that the fibroblast-like cells are mesenchymal in origin and derive from the nonadhering cells. This has been confirmed through proteomics, which has demonstrated the presence of proteins of differentiated cells such as epithelial cells, fibroblasts, keratinocytes, foreskin and epidermis, as well as mesenchymal cells [101] . Furthermore, besides the presence of proteins typically expressed in tissues including fetal liver, brain, heart, pancreas, cochlea and eye, the presence of proteins expressed in human embryonic stem cells has also been detected in www.futuremedicine.com

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amniotic fluid-derived cells, suggesting the presence of undifferentiated multi- or pluripotent cells in the amniotic fluid.

Multipotency of amniotic fluid-derived cells The first indications that the amniotic fluid may contain cells that are not fully differentiated were reported in the early 1990s when small, nucleated cells, which were identified as hematopoietic progenitor cells, were detected [98] . This was followed by the observation that amniotic fluid-derived cells showed characteristics of myogenic cells after culture in rhabdomyosarcoma cell-line-derived supernatant [102] . Later, the multipotency of amniotic fluid-derived cells was demonstrated in vitro through successful differentiation of these cells into osteocytes, adipocytes and fibroblasts [103] . Moreover, amniotic fluid-derived cells have been shown to demonstrate phenotypes similar to BM-MSCs, with the expression of CD90, CD105, CD73 and CD166. In addition, expression of CD49e, CD58, CD54, CD123, CD71 and CD44 was also detected, while hematopoietic markers such as CD45, CD34 and CD14 were absent on these cells. This phenotypic profile was confirmed in other studies, suggesting that the amniotic fluid may be a novel source of MSCs [100,104–107] . Pluripotency of amniotic fluid-derived cells Besides multipotency, pluripotent properties of amniotic fluid-derived cells have also been suggested. In 1999, Mosquera and colleagues demonstrated that these cells have telomerase activity, which is active in all embryonic cell lines and considered as a marker for pluripotency since it prohibits telomere-shortening [108] . Moreover, during the cell maturation process, a decrease in telomere length was observed. The expression of telomerase in these cells has been confirmed using reverse transcriptase (RT)-PCR techniques [70,109] . The pluripotent character of amniotic fluidderived cells is further supported through studies investigating the expression of OCT-4 in these cells. Indeed, a subpopulation of amniotic fluidderived cells was shown to express OCT-4 as well as stem cell factor, vimentin and alkaline phosphatase [99] . Using immunocytochemical analyses, the amount of OCT-4-positive cells was quantified, revealing that 0.1–0.5% of cells expressed this protein. Based on this observation, a two-stage protocol was developed with the aim of developing a method for isolating 282

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MSCs from amniotic fluid-derived cells [100] . Specifically, amniocytes were isolated from 20 patients and cultured in culture-grade dishes. After 5 days, nonadherent cells were collected from the supernatant, expanded and analysed by flow cytometry. The cells expressed SH2, -3 and -4, CD29, CD44, and HLA-A, -B and -C (MHC class I), with partial expression of CD90 and CD105, while expression of CD10, CD11b, CD14, CD34, CD117, HLA-DR, DP, DQ (MHC class II) and epithelial membrane antigen was not detected. Further characterization of these cells using RT-PCR and immuno histochemistry revealed expression of OCT-4 mRNA and OCT-4 protein in all independent cultures. The observation that amniotic fluid contains OCT-4-positive cells has also been confirmed in other studies [110–112] . In accordance with these findings, the presence of OCT-4 was recently demonstrated in the CD117-positive subpopulation of amniotic fluid-derived cells [104] . Moreover, this subpopulation also expressed the embryonic stem cell marker SSEA-4 but lacked the expression of other embryonic-specific surface markers such as SSEA-3 and TRA1–81. Furthermore, the cells were negative for markers of the hematopoietic lineage (CD45) and of hematopoietic stem cells (CD34 and CD133), but expressed CD29, CD44, CD73, -90 and -105, and were also positive for MHC-I and partially positive for MHC-II. In addition, human amniotic fluid-derived cells have also been shown to be capable of activating both an ectopic OCT-4 promoter and an ectopic Rex-1 promoter, making these cells an attractive tool for transfection approaches, as the Rex-1 gene is a transcriptional target of the transcription factor OCT-4 and is expressed in human embryonic stem cells. Although the expression of OCT-4 in amniotic fluid- and membrane-derived cells provides support to the hypothesis that these cells may be pluripotent, given that this marker is necessary for retaining pluripotency and selfrenewal of embryonic stem cells [113,114] , it is important to note that OCT-4 can be detected in some differentiated cells [115] and, furthermore, detection of this marker can also be due to expression of pseudogenes [116] . The potential of amniotic fluid-derived cells to differentiate into cell lines from all three germ layers (ectoderm, endoderm and mesoderm) has been investigated. Differentiation of amniotic fluid-derived cells toward neurogenic lineages was evaluated as an indicator for the potential to form tissues of the ectodermal layer, while the potential of these cells to form tissues of future science group


the mesodermal lineage was mainly assessed through differentiation into osteoblasts, fibroblasts, adipocytes, chondrocytes and endothelial cells. Meanwhile, the capability of these cells to form tissues of the endodermal lineage was demonstrated through differentiation into hepatocytic cells. It has been reported that amniotic fluidderived cells could be induced to differentiate into ectodermal neuron cells when cultured in media containing β-mercaptoethanol and bFGF [100] . By contrast, Prusa et al. did not observe neuronal differentiation of amniotic fluid-derived cells in response to a specific neurogenic differentiation medium [117] . However, when these cells were cultured in standard medium for 3 weeks, the expression of neuronal markers such as CD133, nestin, neurofilament, CNPase, p75, BDNF and NT3 was detected. These authors concluded that neuronal stem or progenitor cells, as well as already-differentiated cells, such as neurons or oligodendrocytes, may all form part of the cell spectrum of native amniotic f luid. Furthermore, these authors suggest that some of the cells described may be amniotic epithelial cells, which have been shown to express neuronal markers in amniotic fluid cell cultures, and that are able to differentiate toward cells with a neuronal-like phenotype that can survive and function in a rat model of Parkinson’s disease [60] . Further investigations using CD117-positive amniotic fluid-derived cells have confirmed the potential of these cells to differentiate toward neuronal lineages [104] . After isolation of the CD117positive subpopulation from cultured amniotic fluid-derived cells using magnetic beads, neurogenic differentiation was induced. Using a two-stage induction procedure, cells were first seeded onto fibronectin-coated plates and incubated in neurogenic induction media, yielding dopaminergic cells. Under these conditions 80% of the CD117-positive cells expressed nestin. In addition, under conditions that favour production of dopaminergic neurons, individual cells with pyramidal morphology were assessed for potassium channels by voltage clamping. These cells demonstrated barium-sensitive potassium channel activity that was consistent with the presence of the GIRK2 channel. Furthermore, when exposed to NGF-containing media, cells acquired the ability to secrete the excitatory neurotransmitter l-glutamine in response to stimulation by potassium ions. Implantation of these cells into the lateral ventricle of a mouse model resulted in smooth cell integration and future science group

Review

survival for up to 2 months without any evidence of deformation of the host tissue or neoplastic processes. However, these results remain the subject of controversial discussion [118] . The potential of amniotic fluid-derived cells to give rise to tissues of the mesodermal lineage has been demonstrated through their ability to differentiate into osteoblast-like cells. After 3 weeks of culture in an osteogenic medium supplemented with dexamethasone, β-glycerol phosphate and ascorbic acid, osteogenesis was demonstrated through von Kossa staining to visualize mineralization of calcium accumulation [103] . The osteogenic potential of amniotic fluid-derived cells has also been confirmed by other groups using similar culture conditions [100,104,107,110,112] . Induction of adipogenesis in amniotic fluidderived cells has been achieved through the use of indomethacin, dexamethasone, methyl3-isobutylxanthine and insulin. Under these conditions, accumulation of lipid vacuoles was observed, thereby proving the in vitro adipogenic differentiation potential of the amniotic fluid-derived cells [103] . This finding was further confirmed by other studies [100,104,107,110,112] . Investigation of the capacity of human amniotic fluid-derived cells to form chondroblasts has demonstrated successful chondrogenic differentiation of these cells after culture in chondrogenic media containing TGF‑b, as recently reported by Kolambkar et al. [119] , while chondrogenic differentiation of these cells has also been shown in animal models [120] . Endothelial cell differentiation has been observed in the CD117-positive [104] and CD133positive [106,107] subpopulations of amniotic fluidderived cells after exposure to VEGF. Besides eNOS and CD141 (thrombomodulin) expression, formation of Weibel-Palade bodies could be detected in the cytoplasm of CD133-positive cells, as indicated by von Willebrand staining. Moreover, when analysed by scanning electron microscopy, cells demonstrated morphologies comparable to heart valve endothelia [106,107] . Currently, little information exists regarding the endodermal differentiation potential of amniotic fluid-derived cells. De Coppi et al. have reported urea production by these cells after 3 weeks of culturing [104] . In this study, CD117-positive cells were seeded onto a collagen sandwich-like layer and biochemically stimulated using a combination of growth factors and cytokines, including monothioglycerol, HGF, oncostatin, dexamethasone, FGF-4 and insulin-transferrin-selenium. During culture, www.futuremedicine.com

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morphological changes were observed whereby cells that were initially elongated adopted a rounded shape typical of hepatocyte-like cells. Moreover, these cells expressed albumin, α-FP, HNF-4α, c-Met and the multidrug-resistance membrane transporter MDR1, thereby suggesting that CD117-positive amniotic fluid-derived cells are able to successfully differentiate toward a cell type of the endodermal lineage.

Amniotic fluid cells as a tool for cell-based therapies Since amniotic fluid has been demonstrated to be a unique pool of fetal cells that bear resemblance to embryonic stem cells, their possible usefulness for developing future cell-based therapy strategies, including tissue engineering and cell transplantation, has been investigated. Based on promising results obtained by Kaviani et al. in 2001, amniotic fluid was proposed as a new cell source for fetal tissue engineering [121] . In this study, amniotic fluid was obtained from pregnant ewes and cultured. Immunocytochemistry revealed expression of fibroblast surface protein, vimentin, smooth muscle actin, CK-8 and -18. When seeded onto a biodegradable synthetic polymer (PGA/P4HB), dense, confluent cell layers surrounding the polymer matrices were observed, suggesting that amniotic fluid-derived cells may be useful for tissue engineering applications. Further studies demonstrated the successful translation of these findings from the animal model to human systems by seeding human amniotic fluid-derived cells onto different polymers (PGA and acellular dermis) [122] . In addition, autologous amniotic fluid cell-based tissue engineered constructs have been successfully used for neonatal repair of the diaphragm [123,124] and for fetal tracheal reconstruction in bovine models [125] . Recently, it has been demonstrated that human amniotic fluid-derived cells have the potential to form fetal-like structures when used for the fabrication of autologous pediatric heart valve leaflets in vitro [106,107] . Here, human amniotic fluid-derived cells were isolated and sorted based on CD133 expression, yielding a noncommitted progenitor cell population with the capacity to differentiate toward endothelial cell lines. Both the CD133-negative and -positive cells were expanded and subsequently analysed. CD133-negative cells expressed CD105, CD90 and CD44 and showed multipotent differentiation potential in vitro when cultured in well-defined osteoblast- and adipocyte284

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inducing media. Meanwhile, CD133-positive cells expressed CD90, CD105 and CD44 but lacked expression of CD34. Exposure of the CD133-positive cells to VEGF resulted in an endothelial cell-like phenotype as demonstrated by eNOS and CD141 expression, as well as formation of Weibel-Palade bodies as detected by vWF staining. A total of 4 weeks after seeding onto biodegradable synthetic polymers (PGA/ P4HB) and exposure to mechanical stimulation, three-layered heart valve tissues, including an endothelium-like surface, were obtained. In vitro functionality tests revealed that the heart valves that had been generated displayed sufficient opening and closing behaviour under low-pressure conditions to suggest that these engineered constructs might function in a lowpressure setting such as in pulmonary valve replacements. Similar results to those described previously were observed for CD133-positive and -negative cells after isolation, cryopreservation and reculturing of these cells. Furthermore, these cells also retained their phenotype and stem cell-like characteristics, as demonstrated by their capacity to differentiate in vitro [107] . This study indicates that cryopreserved amniotic fluid-derived cells are a promising lifelong available autologous fetal stem cell source. The versatility of human amniotic fluid-derived cells for therapeutic applications has been investigated in various animal models. For example, it has been reported that printed, layered scaffold-cell constructs (produced by layer-bylayer printing of human CD117-positive amniotic fluid-derived cells into an alginate/collagen composite gel using thermal inject printing) that were cultured for 1 week in osteogenic differentiation medium mineralized and formed bonelike material when implanted subcutaneously in immunodeficient mice [104] . In addition, it has been shown that human amniotic fluid-derived cells survive and migrate following transplantation into a rat model of cerebral ischemia, as well as in normal rat brains [126] . Furthermore, cell migration toward the borders of lesioned areas was observed in ischemic brains, while cells were seen to spread into multiple regions in normal brains. Additional evidence in support of the regenerative properties of human amniotic fluidderived cells comes from studies in which these cells were transplanted into the injured site of a crushed nerve in a rat model [127,128] . Further studies revealed that concomitant treatment with amniotic fluid-derived cells and granulocytecolony stimulating factor promotes early and future science group


late nerve regeneration [129] . Moreover, it has been suggested that amniotic fluid-derived cells exert beneficial effects on the ischemic brain to an extent comparable with the neuroprotective effect of embryonic neuronal stem cells [130] . Recently, the successful integration of human amniotic fluid-derived cells into murine lungs and their differentiation toward pulmonary lineages after injury have been described [131] . Renal differentiation of human amniotic fluid-derived cells has also been observed in a study in which green fluorescent protein- and Lac-Z-transfected cells were microinjected into murine embryonic kidneys. Monitoring of the cells revealed survival and integration of these cells into the growing organ and contribution to primordial kidney structure, including renal vesicles and C- and S-shaped bodies [132] . While the findings reported above suggest that amniotic fluid-derived cells may be useful for regeneration of renal, pulmonary and neuro nal tissues, studies reported to date regarding the in vivo cardiomyogenic potential of these cells have not yielded such promising results. Chiavegato et al. did not observe integration or differentiation of human CD117-positive amniotic fluid-derived cells into cardiomyocyte-like cells after transplantation into the myocardium of normal, ischemic, immunosuppressed or immunodeficient rats [105] , while the same phenomenon was observed when autologous porcine amniotic fluid-derived cells were injected into an acute myocardial infarction model [133] . Immunological properties of amniotic membrane & fluid-derived cells Given that the placenta forms the interface between the immunologically distinct mother and fetus, it has been proposed that cells from this tissue, including cells from amniotic membrane, have immunomodulatory properties that contribute to maintenance of feto–maternal tolerance during pregnancy. Support for the hypothesis that fetal membranes are nonimmunogenic comes from clinical studies that show that amniotic membrane can be used for treatment of skin wounds, burn injuries and chronic leg ulcers, prevention of tissue adhesion in surgical procedures and ocular surface reconstruction [21–24,27,28,31,34,35] . Moreover, several clinical trials in humans have proven that allogeneic transplantation of amniotic membrane [134–136] or hAECs [137,138] in the absence of immunosuppressive treatment does not induce acute immune rejection. future science group

Review

In vitro studies aimed at understanding the mechanisms underlying the immunomodulatory effects observed after allo- or xenogeneic transplantation of placenta-derived cells show that cells derived from the fetal membranes do not induce an allo- or xenogeneic immune response, and actively suppress T-cell proliferation [49,139] . hAMSCs have been shown to display immunomodulatory effects on mixed lymphocyte reactions (MLRs) both in a direct contact and trans well setting, suggesting that these cells secrete soluble immunomodulatory factors. Recently, in the stromal layer of the amniotic membrane, the presence of two subpopulations that can be distinguished by their differing expression of HLA-DR, CD45 and CD11, and which display either T-cell stimulatory or suppressive effects, has been described, bringing new insight into the immunomodulatory functions of these cells [140] . Furthermore, it has been shown that culture in serum-free conditions allows the selection of a population of amnion-derived multipotent progenitor cells that lack class II antigens and the costimulatory molecules B7–1 and B7–2, but that express HLA-G, the latter of which becomes upregulated after interferon-y treatment. These cells were shown to inhibit peripheral blood mononuclear cell (PBMC) proliferative responses to mitogen, alloantigen and recall antigen, and these effects were shown to be dependent on cell-to-cell contact [141] . In vivo, cells derived from both the human amniotic membrane in toto, as well as the human AE, have been shown to survive for prolonged periods of time after xenogeneic transplantation into immunocompetent animals, including rabbits [142] , rats [37] , guinea pigs [143] and bonnet monkeys [59] . Furthermore, long-term engraftment has been observed after intravenous injection of human amniotic and chorionic cells into newborn swine and rats, with human microchimerism detected in several organs [139] , suggestive of active migration and tolerogenic potential of the xenogeneic cells. Long-term survival of rat amnion-derived cells, with no evidence of immunological rejection or tumor formation, has also been observed after allogeneic in utero transplantation of these cells into the developing rodent brain [86] . Studies investigating the immunological features of human amniotic fluid-derived cells show that these cells weakly express HLA Class I and -G, but not HLA-DR. Production of IL-6 [144,145] has also been observed during culture of these cells, while expression of HLA-DR could be induced after treatment with IFN-γ [145] . www.futuremedicine.com

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A recent study has reported that human amniotic fluid-derived cells can be successfully transplanted into the brains of newborn mice, and can survive in these animals for at least 2 months [104] . However, another report demonstrated rejection of amniotic fluid-derived cells after xenotransplantation in myocardial infarct mouse models after 1 month, suggesting that in vivo use of these cells is hindered by their immunogenic properties. In particular, the expression of costimulatory molecules by these cells seems to play a key role in their rejection [105] . Conclusion & future perspective Data that have been obtained to date regarding cells derived from both the human amniotic membrane and amniotic fluid provide mounting evidence in favour of the possible clinical applicability of these cells in the field of regenerative medicine in the future. To date, many groups have reported that heterogeneous cell populations derived from either the amniotic membrane or amniotic fluid can give rise to cell types of all three germ layers, thereby confirming that these cell populations at least contain progenitor cells, and supporting the

possibility that they may also contain pluripotent stem cells. Currently, this possibility is most strongly supported by the work of Tamagawa and co-workers, through demonstration that clonally derived amniotic cells could be detected in ectodermal, endodermal and mesodermal organ primordia of chimeric embryoid bodies [45] . Although there is great potential for application of amniotic membrane- and fluid-derived cells in regenerative medicine, no clinical experiences have yet been reported, most likely due to the fact that an understanding of the regenerative capacities of these cells and characterization of their properties have only been recently achieved. However, in the future, these cells might play an important role in cell-based strategies as an attractive source of MSCs. Use of these cells to help overcome a lack of umbilical cord bloodderived mesenchymal cells for cotransplantation [103] , based on the demonstration that MSCs enhance the engraftment of umbilical cord blood-derived hematopoietic stem cells [146] , may represent one such example of a possible clinical application for these cells. Moreover, promising preclinical studies suggest that these cells may be a future tool for the treatment of Parkinson’s

Executive summary Cell types Amniotic membrane and amniotic fluid contain mature (epithelial, fibroblast-like mesenchymal cells and macrophage-like cells) and progenitor cells, as demonstrated by expression of pluripotent molecular markers and embryonic stem cell markers, as well as pluripotent differentiation potential and telomerase activity (for amniotic fluid-derived cells). Genetically, cells from both the amniotic membrane and amniotic fluid are of fetal origin. While the amniotic membrane contains cells which are derived exclusively from extraembryonic tissue, the amniotic fluid contains cells shed by both the developing fetus as well as the amniotic membrane. In vitro differentiation potential In vitro differentiation potential toward neurogenic, osteogenic, chondrogenic, adipogenic, hepatogenic and endothelial lineages has been demonstrated for both amniotic membrane-derived cells and amniotic fluid-derived cells. Cardiomyogenic and vasculogenic potential has been demonstrated for amniotic membrane-derived cells. Preclinical application in animal models Functional restoration has been observed after transplantation of amniotic membrane-derived cells into animal models of spinal cord injury, Parkinson’s disease, cerebral artery occlusion, diabetes and myocardial infarction. Besides the feasibility to lead to fetal-like tissues including trachea, diaphragm and heart valves, regenerative properties of amniotic fluidderived cells have been shown in models of neural, lung and renal injuries. Immunological characteristics Amniotic membrane-derived cells display immunomodulatory properties both in vitro and in vivo, while the immunoregulatory properties of amniotic fluid-derived cells are still debatable, as these cells have been shown to be rejected after transplantation in some settings. Both cell sources have been reported to also harbour potential immuno-stimulatory cells. This issue requires further studies. Ethical aspects As a potential source of cells for application in regenerative medicine, the placenta has the advantages of being a natural by-product of birth which is often simply discarded, while harvest of term amniotic membrane and other placental components does not pose any risk to the mother or newborn. Amniotic fluid may represent a valuable autologous stem cell source, particularly for fetal tissue engineering. However, the risk of miscarriage associated with harvest of amniotic fluid, although low (1 out of 200) is still a significant ethical concern. Nevertheless, harvest of amniotic fluid may be a desirable option in cases where the risk–benefit ratio clearly favours the use of these cells. Because amniotic fluid is harvested at an earlier time point in gestation compared with placental tissues, amniotic fluid may contain more immature cells with a higher stem cell potentiality.

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disease, neuronal injuries, lysosomal storage diseases or other neuronal disorders. In addition, amniotic membrane- or amniotic fluid-derived cells might also be useful for tissue engineering based on reports that these cells can form heart valve tissues, tracheal tissues, bone-like tissues and diaphragm-like tissues in vitro. Thus, the unique pools of cells that can be derived from amniotic membrane or amniotic fluid represent promising sources of progenitor/stem cells, which may allow development of native tissues either through tissue engineering or cell transplantation. In addition, the possibility of banking these cells is likely to extend their applicability to allow their use during adult life as well as during the neonatal period, while use of amniotic fluid-derived cells may also provide a feasible treatment option in cases where cells are required during fetal development or immediately after birth. In addition, the low immunogenicity and immunomodulatory characteristics of these cells also opens the way for their use in allogeneic transplantation settings. An interesting question that is emerging in the field of research into amniotic cells concerns whether it is their differentiation potential Bibliography

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